A Semi-Selective Agar Medium to Detect the Presence of ...Fitopatol. bras. 30(5), set - out 2005 489...

Fitopatol. bras. 30(5), set - out 2005 489

A Semi-Selective Agar Medium to Detect the Presence ofXanthomonas axonopodis pv. malvacearum in Naturally Infected

Cotton SeedYeshwant R. Mehta1, Cleide Bomfeti2 & Viviani Bolognini2

1Laboratório de Patologia de Sementes, Área de Proteção de Plantas, Instituto Agronômico do Paraná-IAPAR,Cx. Postal 481, Londrina, PR, Brasil, e-mail: [email protected]; 2Bolsista FACUAL

(Accepted for publication on 23/06/2005)

Corresponding Author: Yeshwant R. Mehta

MEHTA, Y.R., BOMFETI, C. & BOLOGNINI, V. A semi-selective agar medium to detect the presence of Xanthomonasaxonopodis pv. malvacearum in naturally infected cotton seed. Fitopatologia Brasileira 30:489-496. 2005.

ABSTRACTA semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam)

in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selectivemedium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibioticsin different concentrations were tested alone or in combination with others. The final composition of the semi-selectiveagar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes andfavour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid.The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on thesemi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selectivemedium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was notalways correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detectthe presence of Xam in naturally infected cotton seed.

Additional keywords: Gossypium hirsutum, black arm of cotton, angular leaf spot.

RESUMOMeio semi-selectivo para detectar a presença de Xanthomonas axonopodis pv. malvacearum em sementes de algodoeironaturalmente infetadas

Um meio de cultura semi-seletivo foi desenvolvido para detectar Xanthomonas axonopodis pv. malvacearum (Xam)em sementes do algodoeiro (Gossypium hirsutum). O meio básico foi peptona-sacarose-ágar (PSA). Os critérios para omeio semi-seletivo foram; as características típicas das colônias de Xam e sua patogenicidade em algodoeiro. Váriosfungicidas sistêmicos e antibióticos foram testados sozinhos ou em combinação com outros em diferentes concentrações. Acomposição final do meio semi-seletivo foi estabelecida após várias tentativas no sentido de inibir a maioria dos fungos ebactérias saprofíticas e favorecer o desenvolvimento de Xam. O meio contém: PSA + ciclohexamida, cefalexina, pencycuron,triadimenol e tolylfluanid. A bactéria foi recuperada de sementes naturalmente infetadas através do plaqueamento direto de2.000 sementes externamente desinfestadas em meio semi-seletivo. A recuperação do patógeno de tecidos homogenizadosda folha infetada e a recuperação em plaqueamento por diluição, em meio semi-seletivo e em agar nutriente, foramcomparáveis. Entre os três métodos de detecção, o meio semi-seletivo foi o mais preciso e confiável. O nível da severidadede infecção em campo não era sempre correlacionado com o nível de infecção nas sementes. Este é o primeiro relato demeio semi-seletivo para detectar a presença de Xam em sementes do algodoeiro.

Palavras-chave adicionais: Gossypium hirsutum, mancha angular.

INTRODUCTION

Angular leaf spot caused by Xanthomonasaxonopodis pv. malvacearum (Xam) [Sin. X. campestris pv.malvacearum (Smith) Dye)] is one of the most importantdiseases of tetraploid cotton (Gossypium hirsutum L. and G.barbadense L.) in many countries, including Brazil. Thedisease can cause heavy yield losses depending on the yearand the cultivar. Cotton seed is considered to be an importanttransmission vehicle of Xam and a source of primary

inoculum (Bain, 1939; Brinkerhoff & Hunter, 1963; Hunter& Brinkerhoff, 1964; Mohan, 1983a, 1983b). Tarr (1961)and Brinkerhoff & Hunter (1963) reported that internallyinfected seed between 0.017 and 2.0% resulted in severelosses in commercial cotton fields in Sudan and in the USA.So far, in Brazil, there is no regulation, either for fieldinspections for angular leaf spot or for laboratory testing, ofseeds infected with the pathogen. As a result, the disease ison the increase in the States of Paraná, São Paulo, Goiás,and Mato Grosso, and severe epiphytotics of the disease

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Y.R. Mehta et al.

occurred in 2001, 2002 and 2003 in these states. Currently,the Brazilian Ministry of Agriculture is considering theadoption of rules and regulations requiring field inspectionsfor the presence of angular leaf spot disease. A zero tolerancefor fields infested with the disease is being proposed. Anyseed production field with the disease, irrespective of thelevel of infection, will be destined for commercial cottonand not for seed production. This stringent regulation maycreate seed shortages and other problems. The year 2003was very favourable for angular leaf spot, and in the Stateof Mato Grosso, there was not a single field which wascompletely free from the disease. Thus, the success of a seedhealth certification program, as in other cases (Mohan &Schaad, 1987), would depend on the availability of a reliablemethod to detect Xam in naturally infected seeds.

Several semi-selective agar media were reported forphytopathogenic bacteria like Corynebacterium spp.(Clavibacter), Erwinia spp., Pseudomonas spp. andXanthomonas spp. (Kado & Heskett, 1970), Xanthomonastranslucens pv. undulosa (Schaad & Forster, 1985), X.axonopodis pv. phaseoli (Clafin et al., 1987), andPseudomonas syringae pv. syringae (Mohan & Schaad,1987). In Israel, a method to detect the pathogen in the seedlot, but not quantitatively, was reported by Halfon-Meiri &Volcani (1977). A semi-selective medium to detect and toquantify the presence of Xam in cotton seed has not beenreported. Because of the lack of an appropriate, sensitive,selective medium, research on control measures includingmethods to erradicate the bacteria from the seed is hampered.A preliminary report about semi-selective medium has beenpublished (Mehta & Bolognini, 2003). This paper describesa semi-selective agar medium and compares other methodsto detect the presence and longevity of Xam in naturallyinfected cotton seed.

MATERIALS AND METHODS

Development of semi-selective agar mediumSome of the semi-selective media reported for

xanthomonads (Schaad & Forster, 1985; Claflin et al., 1987)were evaluated in preliminary tests. Some systemic fungicideswere tested alone or in combination with others in differentconcentrations. Some of the antibiotics were also evaluatedby paper disc method (Randhawa & Schaad, 1984). Thebasic medium was peptone-sucrose-agar (PSA), containing0.35 g Ca (NO3), 0.35 g FeSO4, 1.4 g Na2HPO4, 3.5 gpeptone, 14.0 g sucrose, 10.5 g bacto-agar, 700 ml distilledwater, pH 6.8. Criteria for the semi-selective medium werethe typical colony characters of Xam and its pathogenicityon cotton. The final composition of the semi-selective agarmedium was established so as to inhibit most of the fungaland bacterial saprophytes and to favour the isolation of Xam.It contained: PSA + cycloheximide (Sigma) 100 mg (2 mlof a stock solution of 500 mg in 10 ml of 75% alcohol);cephalexin (Sigma) 10 mg (1 ml of a stock solution of 100mg in 10 ml of 75% alcohol); pencycuron 0.05 g (Monceren

PM, Bayer S.A.); triadimenol 1 ml (Baytan SC, Bayer S.A.);tolylfluanid (Euparen 500PM, Bayer S.A.) (1 ml of a stocksolution of 0.125 g in 10 ml of sterilized water). Thefungicides pencycuron, triadimenol and tolylfluanid wereefficient in inhibiting the development of most of the seed-borne fungal saprophytes. Tolylfluanid as a seed dressingfungicide is also known to control Rhizoctonia solani Kühnin cotton seedlings (Goulart & Ferraz, 2001). Antibioticsand fungicides were added, after the medium was autoclaved,in the order presented above. In all the tests, young cultureof Xam was streaked onto two others containing PSA andtwo plates containing semi-selective medium and maintainedas controls to ensure that in none of the tests Xam wasinhibited by the semi-selective medium.

Plating efficiencyPlating efficiency was determined in comparison with

Difco nutrient agar (NA) as a standard medium and wasevaluated by colony counts at 10 fold serial dilutions (10-4

,10-5, 10-6, 10-7), using young cultures of 13 strains ofXanthomonas sp. in three replicates. Initial cell suspensionwas adjusted to an absorbance of 0.1 at 640 nm using aspectrophotometer. One hundred microlite of each dilutedsuspension was distributed on each Petri dish containingeither NA or semi-selective medium. Dilutions yielding over300 colonies per plate were not considered for counting.The mean percentage of recovery of the colony forming units(cfu) were compared with NA. The percentage of platingefficiency was calculated in relation to the cfu developed onNA, three days after incubation at 24 oC. Recovery of Xamfrom homogenized diseased leaf tissues was also verifiedon both NA and semi-selective media.

Seed samplesSamples were obtained from six seed lots belonging

to G. hirsutum (three of cv. ITA 90, one of cv. Fabrika andtwo of cv. Makina) from a 2001 harvest in Mato Grosso offields producing seeds with angular leaf spot disease (Table3). The level of disease incidence in these fields was notknown. After delinting with concentrated sulfuric acid(Brinkerhoff & Hunter, 1963) the samples were gentlywashed three times with tap water, air-dried on filter paperand stored at 5 oC for further use.

Seed testing assaysTo detect the presence of seed infection in six seed

lots, the following methods were used. For all pathogenicitytests, first leaves of 30-35-day-old plants of cvs. Ita 90 andSaturno were inoculated (three plants per cultivar) using atooth pick method (Cia et al., 1973). Inoculated plants wereincubated in a dew chamber for 48 h and later placed on thegreenhouse bench and examined periodically for symptomexpression.

Seed washing. Seeds (200 g) were disinfested with70% ethyl alcohol for 1 min, immersed 4 min in sodiumhypoclorite (2.5% of NaClO), followed by three quick

A semi-selective agar medium to detect the presence of...


washings with sterilized water. The disinfested seeds werefinally washed in 300 ml of saline water (0.5% NaCl) for24 h in a mechanical shaker in three replicates. Thesuspension was filtered through Whatman filter paper No.1, centrifuged for 10 min at 10,000 rpm. The pellet wassuspended in 5 ml of distilled water and used forpathogenicity tests in the greenhouse;

Direct seed plating. Two thousand seeds weresurface disinfected as mentioned earlier for seed washing,but instead of washing again for 24 h, were placed in Petriplates (ten seeds per plate) each containing 15 ml of semi-selective medium. Plates were examined periodically sevendays after incubation. Within seven-12 days after incubation,typical colonies of Xam growing around the seeds wereobserved and were tested directly for pathogenicity. Theidentification of the Xam colonies was based onmorphological characteristics like size, colour andconsistency which were also confirmed by streaking ontoPSA plates. After 12 days no new colonies of Xam wereobserved.

Growing-on-test. Two thousand seeds of each seedlot were sown in plastic trays (20 x 35 cm) containingunsterilized soil in the greenhouse (100 seeds per tray), trayswere irrigated once a day and the plants were examined forsymptom development 15-35 days after sowing. The averagegreenhouse temperatures during the experimental periodvaried between 18 and 26 oC, and the relative humiditybetween 60 and 85%. Care was taken to avoid countingplants with secondary infections. For this purpose, plantswere examined daily and the infected ones were markedwithin the first 25-30 days after sowing. Although secondaryinfection was rare, it was normally identified as a few water-soaked angular spots on the first or second leaves on plantswith no other symptoms. Invariably such plants weresurrounded by the initially infected plants.

LongevityLongevity of Xam in two seed lots was studied by

plating 2,000 surface desinfested seeds periodically onto thesemi-selective medium using the same procedure as statedfor direct seed plating. The coefficient of correlation betweenthe period of seed storage and counting the number ofbacterial colonies was evaluated by regression analysis usingthe “Cricket graph” program, version 1.01 of Macintosh.

Correlation between disease severity in the field and seedinfection

The disease severity levels of angular leaf spot in thefield (estimated as the percentage of leaf area infected) werecorrelated with the level of infection in the seed by platingupto a total of 4,800 seeds of some samples onto the semi-selective medium. For this purpose, the disease severity scaleof 0-4 for field infection was used, where, 0= no disease,1=trace - less than 5% of the leaf area infected (LAI); 2=low (<25% LAI); 3=medium (<50% LAI); 4=severe (>50%LAI).

RESULTS

Plating efficiencyIn the dilution plating, the percentage of Xam

recovery on semi-selective agar medium compared well withNA. Colonies were recovered in both media even at a veryhigh dilution indicating the high sensitivity of the semi-selective medium to detect the pathogen. (Table 1, Figure1). The colonies of Xam grew faster and were larger onsemi-selective medium than on NA. Plating efficiencies (%mean recovery) on semi-selective medium varied between zeroand 187%. Whereas the semi-selective medium favoured goodrecovery for all the strains of Xanthomonas sp. from cotton,the recovery of one strain of X. axonopodis pv. phaseoli(Smith) Dye was zero. The semi-selective mediuminhibited the growth of X. axonopodis pv. phaseoli duringthe first three days after incubation (Table 1). Few andvery small colonies of this strain developed five days afterincubation and hence were not considered for analysis.Amongst the xanthomonads the percentage recovery of X.axonopodis pv. citri was one of the highest on semi-selectivemedium.

Recovery of Xam from homogenised diseased leaftissues was similar on both NA and semi-selective mediumindicating the non-toxicity of the semi-selective medium(Figure 2).

Seed washingWhen suspensions of seed washings were inoculated

on susceptible cotton plants, typical disease symptoms werereproduced 12 days after inoculation in seed sample Nos. 5and 6 in all the three replications (Table 2).

Direct seed platingGrowth of most of the saprophytic bacteria and fungi

was inhibited by the semi-selective agar medium and by thedisinfestation of the seeds by alcohol and by sodiumhypoclorite. Since the pathogen is internally seed-borne,detection of Xam was not affected by disinfestation asevidenced by some preliminary tests. The addition ofgentamycin inhibited the development of most of the fungiand bacteria (Schaad & Foster, 1985), but it was toxic toXam. Susbstitution of gentamycin by pencycuron,triadimenol and tolylfluanid, inhibited the development ofmost of the fungi but at the same time did not affect thedevelopment of Xam. Addition of kasugamycin did notimprove the medium. It was non toxic to Xam as well as toother saprophytic bacteria. Laboratory tests indicated thatsome of the saprophytic bacteria associated with the cottonseed were not antagonistic to Xam. The typical colonies ofXam growing around the seed were slightly yellowish,smooth, convex and glistening (Figure 3). The identity ofsuch colonies was always confirmed by streaking onto thePSA medium and by the pathogenicity test in the greenhouse.Out of six seed lots two were free from the bacteria. Thesemi-selective medium could detect a very small percentage


Y.R. Mehta et al.

NA

SSM

of infection (0.05%), which otherwise was not detected byany other method (Table 2).

Growing-on-testThe pathogen transmission by growing-on-test in one

seed lot was demonstrated under greenhouse conditions(Table 2). Twenty five to 30 days after sowing, typical watersoaked translucent angular leaf spot symptoms developedon the first leaves of cv. Makina which in severe cases causeddeath of the seedling. The identity of the pathogen was

confirmed by isolation and by pathogenicity tests in theglasshouse. As mentioned earlier, failure to demonstrate thetransmission of the bacteria in other seed lots by growing-on-test was possibly due to a relatively low level of seedinfection. Besides, an inapropriate temperature and humidityregime during the experimental period may inhibit thedevelopment of typical angular leaf spot symptoms, andconsequently, a particular seed lot may be wrongly identifiedas free from Xam. The exact temperature and humidityregime for seed transmission is not well understood.

TABLE 1 - Number of strains of Xanthomonas spp. on semi-selective agar medium compared togrowth on Difco nutrient Agar

*Xan=Xanthomonas. All strains belong to the culture collection of IAPAR, Londrina;**(Number of colonies recovered on semi-selective agar medium x 100)/number of colonies on Difco nutrient

agar). Calculated from the mean number of colonies per plate in three replications per strain. Columns with thesame letter do not differ from each other according to Tukey at 5% (p=0.05). Xanthomonas strains frompassion fruit, citrus, and beans, were collected by Rui Perreira Leite, IAPAR, Londrina, PR.

FIG. 1 - Comparison of colony forming units of Xanthomonas axonopodis pv. malvacearum in dilution plating(from left to right - 10-4,10-5,10-6,10-7) between Difco nutrient agar (upper plates) and on semi-selective agarmedium (lower plates), three days after incubation.

____________________________________________________________

Strain/Pathovar* Host/State Mean recovery(%)**____________________________________________________________

Xan. 13403/malvacearum Cotton/Mato Grosso 66 ed

Xan. 13355/malvacearum Cotton/Mato Grosso 86 ced

Xan. 13360/malvacearum Cotton/Mato Grosso 100 cbd

Xan. 13362/malvacearum Cotton/Mato Grosso 87 ced



Xan. 13856/malvacearum Cotton/Mato Grosso 65 ed

Xan. 138058/malvacearum C otton/Mato Grosso 158 cb


Xan. 6667/undulosa Wheat ( L.) ParanáTriticum aestivum / 57 ed

Xan. 12917/citri Citrus ( spp.)/ParanáCitrus 187 a

Xan. 3701/phaseoli Beans ( spp.)/ParanáPhaseolus 0 e

Xan. 11370/passiflora Passionfruit ( Sims)/ParanáPassiflora edulis 56 ed____________________________________________________________

____________________________________________________________



FIG. 2 - Recovery of Xanthomonas axonopodis pv. malvacearumfrom homogenised diseased cotton (Gossypium hirsutum) leaftissues on Difco nutrient agar (left) and on semi-selective agarmedium (right), three days after isolation.

LongevityBy using the semi-selective medium it was possible

to study the longevity of Xam in two naturally infected cottonseed lots. The viability of bacteria started declining gradually12 months after harvest and reached the lowest level after23-26 months of storage (Table 3). The reduction in thelevel of seed infection was inversely proportional to theincrease in storage period. For the seed lots No. 5 and 6, thecoefficient of correlation (r) was 0.890 and 0.975,respectively. Unfortunately, there is no informationregarding whether there was a decline or not in the level ofseed infection during the first 12 months after harvest, sincethe semi-selective medium was developed after this period.The results showed that the bacteria can survive in theinfected seed for a period of at least 28 months, especiallywhen the seed is stored at 5 oC. The longevity of Xam indried leaves preserved under laboratory conditions for sevenyears and in the seed for 56 months was demonstrated bySchnathorst (1964) and Hunter & Brinkerhoff (1964),respectively. It is also known that X. campestris pv. undulosa

TABLE 2 - Detection of Xanthomonas axonopodis pv. malvacearum (Xam) in six naturally infected commercial cotton (Gossypiumhirsutum) seed lots by three different methods

*See text for details of the three methods of detection.**Number (percentage of infected seed with Xam).***After seed washing the pellet containing Xam produced typical symptoms of the disease 12 days after inoculation. Delinting was done using concentrated

sulphuric acid (Brinkerhoff & Hunter, 1963).

FIG. 3 - Typical colony growth of Xanthomonas axonopodis pv.malvacearum indicated by an arrow, recovered on semi-selectiveagar medium from naturally infected cotton (Gossypium hirsutum)seed, eight days after incubation at 24o C.

Hagb of wheat, for example, can survive in the infected seedfor a period of over four years (Mehta, 1990; Mehta & Bassoi,1993; Bragaard et al., 1993). Further research is needed toverify the longevity of Xam in the infected seed at the normalstorage conditions (about 24 oC) commercially practiced inBrazil.

Correlation between disease severity in the field and seedinfection

Results indicate that the disease severity levels ofangular leaf spot in the field did not always correlated withthe level of infection in the seed (Table 4).

DISCUSSION

In general, the semi-selective medium showed goodplating efficiency for the strains of X. axonopodis pv.

Seed infected with Xam

Method of detect ion*Seed lot and cotton cultivarOrigin

Location/State/YearSeed washing

Seed plating on semi-selective medium**

Growing- on-Test

Fabrika Rondonó polis,MT/2001 Negative 4 (0.20%) 0

Ita 90 A Serra de Petrolina, MT/2001 Negative 0 0

Makina Rondonó polis, MT/2001 Negative 1 (0.05%) 0

Ita 90 B (treated with unknown fungicide) Rondonó polis (Salles), MT/2001 Negative 0 0

ITA 90 C Itiquira, MT/2001 Positive*** 21 (1.05%) No data due to

short of seed

Makina Costa Rica, MT/2001 Positive*** 20 (1.00%) 31 (1.55%)


Y.R. Mehta et al.

malvacearum as compared to other xanthomonads. Amongthe three detection methods tested, the semi-selective

medium was found to be the most reliable for quantitativeassays. The identity of Xam colonies appearing around theinfected seeds on the semi-selective agar medium wasconfirmed by pathogenicity tests, which are considereddesirable since some of the colonies of Xam may loose theirpathogenicity, or else they may still be confused with someof the yellow pigmented saprophytic bacteria. Alternatively,identification of Xam colonies may also be confirmed bymorphological characteristics by streaking onto the PSAmedium and comparing with the type culture colonies ofXam. Our experience shows that the colonies, onceconfirmed by streaking onto the PSA medium, were alwayspathogenic to cotton. Pathogenicity tests may be dispensableif identification is coupled with molecular analysis using ahighly specific DNA probe. Nonetheless, the semi-selectiveagar medium presented here is practical and can be usedfor screening large number of seed samples. The mediumcan also be used for quarantine purposes.

Normally, the percentage of seed infection by Xamis very low. A relatively low percentage of seed infection,however, would be sufficient to create a severe epidemic ofthe disease under favourable field conditions (Brinkerhoff& Hunter, 1963). Considering the low percentage of seedinfection and the possible establishment of a zero tolerancelevel for seed infection by the Ministry of Agriculture ofBrazil, we suggest that 2,000 seeds per seed lot should beexamined.

Although the pathogen can survive in seed and involunteer cotton plants, no secondary or collateral hosts areknown. The bacterium like most xanthomonads, is sensitiveto drying and excessive heat, and is rapidly destroyed when

TABLE 3 - Longevity of Xanthomonas axonopodis pv. malvacearum (Xam) in naturallyinfected cotton (Gossypium hirsutum) seeds stored at 5o C soon after harvest

*Seed samples originated from fields with angular leaf spot from the State of Mato Grosso, and soonafter harvest in July, 2001, stored at 5 oC. For each analysis 2,000 surface sterilised seeds were placedon Petri dishes containing semi-selective medium. Typical Xam colonies were observed eight-ten daysafter incubation at 24 oC in dark. (-) indicates lack of data. The coefficient of correlation (r) betweenthe increase in storage period and the number of colonies of Xam, for cv. Ita 90 and for cv. Makina was0.890 and 0.975 respectively.

TABLE 4 - Relationship between angular leaf spot severity inthe field and the percentage of infected cotton (Gossypiumhirsutum) seeds with Xanthomonas axonopodis pv. malvacearum(Xam) determined by using the semi-selective medium

*Seed samples originated from different fields or without angular leaf spotfrom the State of Mato Grosso, with the exception of the Breeder´s linewhich was from the State of Paraná. Soon after harvest in June, 2003, theseeds were disinfested and were analised using semi-selective medium.Typical Xam colonies growing around the infected seeds were observed 8-12 days after incubation at 24 oC in dark.

**Disease severity scale of 0-4 for field infection was used, where, 0= nodisease, 1=trace - less than 5% of the leaf area infected (LAI); 2= low(<25% LAI); 3=medium (<50% LAI); 4=severe (>50% LAI).

***For the first five seed lots 4,800 seeds were analysed in two replicationsof 2,400 seeds each. Due to the limited seed of the rest of the seed lots,only 800 seeds were analysed.

Date of seed analysis/ Number of seeds which anabled the recovery of Xamrecovered onmonths after harvest

on semi - selective medium from two seed lots*

Seed lot - cv. Ita 90 Seed lot - cv. Makina

August 5, 2002/12 21 20

October, 7, 2002/14 - 13

February 24, 2002/18 - 11

March 19, 2003/19 - 6

April 2, 2003/20 5 -

May 12, 2003/21 2 -

Ju ne 5, 2003/22 - 1

July 5, 2003/23 2 0

October 1, 2003/26 4 - (Short of seed)

December 5, 2003/28 4 - (Short of seed)

CottonCultivar*

Diseaseseverity

No. of seedsAverage No. %

in the field**

analysed usingof infected infectedsemi - selective

seeds seedmedium***

3 4,800 0 0

Saturno 3 4,800 9 0.38

ITA 90 - D 2 4,800 0 0

ITA 90 - E 3 4,800 1 0.04

ITA 90 - F 4 4,800 5 0.21

Alcapi 0 800 0 0

Alcapi 3 800 4 0.5

Delta Opal 0 800 0 0

STO 474 1 800 0 0

Saturno 2 800 0 0

Breder’s line



crop residue is buried soon after the cotton harvest (Arnold& Arnold, 1961; Brinkerhoff & Fink, 1964; Mehta, 1993).Such conditions prevail especially in the State of MatoGrosso, and thus reduce the possibility of survival of thepathogen in infected cotton plant residue. Althoughlongevity and the role of inoculum present in crop residueunder Brazilian conditions are not well studied, our resultsshow that the seed could be an important source of primaryinfection of angular leaf spot in Brazil.

The disease severity levels of angular leaf spot in thefield were not always correlated with the level of infectionin the seed, even when the test was repeated and a total of4,800 seeds of some samples were plated onto the semi-selective medium (Table 4). Similar results were obtainedby Mohan & Schaad (1987) while working with bean blightbacteria. Because of the limited sample size, only 800 seedswere tested of some seed lots. Correlation between the levelof field infestation and the level of seed infection woulddepend on the climatic conditions between the boll formationand boll opening. Future research using a large number ofseed samples is considered essential to improve ourunderstanding of the correlation between field infection andseed infection. Nonetheless, results indicate that the seedproduction fields infested with angular leaf spot disease cannot be simply condemned. To the contrary, it would beworthwhile to adopt a zero tolerance level for seed infectionbut not for field infestation, especially now when the semi-selective medium has been made available. Besides, fieldinspections are not always very reliable and nor foolproof(Mohan & Schaad, 1987).

This is the first report of a semi-selective agarmedium to detect the presence of Xam in commercial cottonseed. The development of such a medium would certainlyprovoke furture investigations into the establishment ofappropriate methods designed to erradicate the pathogenfrom the cotton seed. The use of semi-selective mediumwould permit planting of only the healthy seeds forcommercial cultivation. Along with healthy seeds the useof resistant cultivars would effectively control the angularspot disease of cotton.

ACKNOWLEDGMENTS

The present research was sponsored by Facual –Fundação Apoio a Cultura de Algodão, MT, Brazil. Thanksare due to Paulo Aguiar, Fabiano V. Sequeri and Tatiane S.Zambosi for supplying part of the seed material from theState of Mato Grosso, and for technical assistance providedby J.A. Oliveira and Edna Marcolini.

LITERATURE CITED

ARNOLD, M.H. & ARNOLD, K.M. Bacterial blight ofcotton:trash-borne infection. Empire Cotton Growing Review38:258-270. 1961.

BAIN, D.C. Effect of sulphuric-acid treatment on fungi andbacteria present on cotton seed from diseased bolls. Phytopathology29:879-884. 1939.BRAGAARD, C., MEHTA, Y.R. & MARAITE, H. Serodiagnosticassays vs. the routine techniques to detect Xanthomonascampestris pv. undulosa in wheat seeds. Fitopatologia Brasileira18:42-50. 1993.BRINKERHOFF, L.A. & FINK, G.B. Survival and infectivity ofXanthomonas malvacearum in cotton plant debris and soil.Phytopathology 54:1198-1201. 1964.BRINKERHOFF, L.A. & HUNTER, R.E. Internally infected seedas a source of inoculum for the primary cycle of bacterial blight ofcotton. Phytopathology 53:1397-1401. 1963.CIA, E., BALMER, E., FERRAZ, C.A.M., GRIDI-PAPP, I.L. &PARADELA FILHO, O. Variabilidade de Xanthomonasmalvacearum (E.F. Smith) Dowson, no Estado de São Paulo. Anaisda Escola Superior da Agricultura “Luiz de Queiroz” 30:457-463.1973.CLAFLIN, L.E., VIDABER, A.K. & SASSER, M. MXP, a semi-selective medium for Xanthomonas campestris pv. phaseoli.Phytopathology 77:730-734. 1987.GOULART, A.C.P. & FERRAZ, D.M. Efeito do tratamento desementes de algodão com fungicidas no controle do tombamentode plântulas causado por Rhizoctonia solani. Anais, 3º CongressoBrasileiro de Algodão, Campo Grande, MS, 2001. pp.560-563.HALFON-MEIRI, A. & VOLCANI, Z. A combined method fordetecting Colletotrichum gossypii and Xanthomonas malvacearumin cotton seed. Seed Science & Technology 5:129-139. 1977.HUNTER, R.E. & BRINKERHOFF, L. A. Longivity ofXanthomonas malvacearum on and in cotton seed. Phytopathology54:617. 1964.KADO, C.I. & HESKETT, M.G. Selective media for isolation ofAgrobacterium, Corynebacterium, Erwinia, Pseudomonas andXanthomonas. Phytopathology 60:969-976. 1970.MEHTA, Y.R. Management of Xanthomonas campestris pv.undulosa through cereal seed testing. Seed Science andTechnology, 18:467-476. 1990.MEHTA, Y.R. Manejo integrado de enfermedades del trigo.Imprenta Landivar, Santa Cruz de la Sierra, Bolivia. 1993.MEHTA, Y.R. & BASSOI, M.C. Guazatine Plus as a seedtreatment bactericide to eradicate Xanthomonas campestris pv.undulosa from wheat seeds. Seed Science and Technology 21:9-24. 1993.MEHTA, Y.R. & BOLOGNINI, V. Meio semi-seletivo paradetectar a presença de Xanthomonas axonopodis pv. malvacearumnas sementes do algodoeiro. Fitopatologia Brasileira 28:240. 2003(Abstract).MOHAN, S.K. A survey of methods for detection of Xanthomonascampestris pv. malvacearum in cotton seed. Seed Science andTechnology 11:569-571. 1983a.MOHAN, S.K. Seed transmission and epidemiology ofXanthomonas campestris pv. malvacearum. Seed Science andTechnology 11:859-865. 1983b.MOHAN, S.K. & SCHAAD, N.W. An improved agar plating assayfor detecting Pseudomonas syringae pv. syringae and P. s. pv.phaseolicola in contaminated bean seed. Phytopathology 77:1390-1395. 1987.


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SCHNATHORST, W.C. Longivity of Xanthomonas malvacearumin dried cotton plants and its significance in dissemination of thepathogen on seed. Phytopathology 54:1009-1011. 1964.TARR, A.S.J. Seed treatment against blackarm disease of cottonin the Sudan. V. Results with seed carrying light and moderateinfection. Empire Cotton Growing Review 38:30-35. 1961.

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